Flow cytometers are commonly employed in the medical industry to analyze particles in a patient's body fluid (e.g., blood cells) as an adjunct to the diagnosis and treatment of disease. As a non-limiting example, in the course of chemotherapy treatment, such instruments may be used to sort and collect healthy blood cells (stem cells) from a quantity of blood that has been removed from a patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is then reinjected back into the patient, to facilitate migration and healthy blood cell reproduction.
For this purpose, as illustrated in the cytometer system diagram of FIG. 1, particles 11 to be analyzed, such as cells of a centrifuged blood sample stored in a container 11, are injected into a (pressurized) continuous or uninterrupted stream of carrier fluid (e.g., saline) 12. This carrier fluid stream is directed along a flow channel 13 of a fluid flow chamber or cell 14. The fluid flow channel 13 is intersected at a location 15 by an output beam 16 emitted by an optical illumination subsystem, such as one or more lasers 17. Located optically in the path of the laser output beam 16 after its being intercepted by the carrier fluid stream are one or more photodetectors of a photodetector subsystem 20. The photodetector subsystem 20 is positioned to receive light modulated by the contents of (particles/cells within) the fluid stream, including light reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell.
In order to avoid confusion as to which photodetector output signal is representative of which illuminated cell, the fluid flow channel 13 through the cytometer flow chamber is configured and sized to pass the particles or cells only one cell at the time through the intersection location 15 with the laser's output beam 16. As a consequence, as shown in the timing diagram of FIG. 2, as output signals from the photodetector subsystem 20 are modulated by particles transported by the carrier fluid stream, each modulation signal, such as that shown at 21 and occurring at a time t0 in the timing diagram of FIG. 2, can be associated with an individual cell. If the output of the photodetector subsystem 20 satisfies prescribed `sort` criteria associated with one or more parameters of a desired cell, it is used to control the sorting of a droplet 23 of carrier fluid containing that cell by an electrostatic droplet sorter 24 located downstream of an exit port or aperture 18 of the flow chamber.
The carrier fluid stream is converted into individual droplets by an acoustically (e.g., piezoelectric transducer) driven droplet generator 27, which is coupled to the fluid flow chamber. The individual droplets do not form immediately at the exit port 18 of the fluid flow chamber, but proceed as an interconnected droplet stream 22 and break off at a location 25 downstream of the chamber exit port. Also, there is a `sort` delay or interval of time t01 between the time t0 that the cell passes through the laser beam intersection location 15 and a subsequent time t1 at which the last attached portion of the carrier fluid stream containing that cell actually physically separates from the carrier fluid stream as a distinct droplet 23 in a stream or sequence of droplets traveling along a vertical travel path 26.
The location 25 at which the droplets form downstream of the flow chamber exit port 18 is adjusted by varying the parameters of the droplet generator drive signal. The rate at which droplets are formed is governed by the frequency of the acoustic drive signal, and the droplets become synchronized with the frequency of the piezo vibration of the droplet generator 27. As a non-limiting example, the acoustic drive frequency applied to the droplet generator 27 may be on the order of from four to one hundred Khz, at a fluid pressure on the order of from three to seventy psi.
The photodetector output is typically digitized and then analyzed by a cell type mapping or identification algorithm executed by an associated supervisory control processor of the cytometer's control workstation 50. Based upon this analysis, the control processor supplies control signals to a charging and deflection control circuit 52 of the droplet sorter 24 to sort or abort the droplet.
In order to controllably sort an individual droplet 23 that breaks off or separates from the fluid stream exiting the flow chamber's outlet port 18, the droplet sorter 24 employs an electrostatic charging collar 31 surrounding the travel path 26 of the droplet sequence. Charging collar 31 may comprise a metallic cylinder that is located so as to surround the location along the droplet sequence travel path 26 where the individual droplets 23 separate from the fluid stream, and is typically several droplets in length. The charging collar 31 is positioned vertically downstream of the fluid chamber exit port 18 and upstream of an associated set of electrostatic (opposite polarity, high voltage) deflection plates 33 and 35 between which the stream of charged droplets 23 pass as they travel downwardly and are either sorted along a sort path 36 into a sorted droplet collection container 41, or allowed to pass unsorted along travel path 26 into an aborted or discarded waste container 43.
Under the control of the cell analysis and sorting routine executed by the system workstation 50, a prescribed charging voltage pulse 32 of a duration t12 is selectively applied to the charging collar 31 at time t1, i.e. at the end of the sort delay t01, and terminating at time t2 at the end of the pulse duration interval t12, thereby charging a droplet 23C that should contain the cell to be sorted. As the selectively charged droplet 23C passes between the two opposite polarity high voltage deflection plates 33 and 35, it is attracted to the plate with the opposite charge, while being simultaneously repelled by the plate with the same or like charge. This electrostatic steering action directs the charged droplet 23C along a deflected travel path 36 on one side of the main droplet travel path 26, and into the sorted droplet collection container 41.
As described above, for any given cell or particle interest within the fluid stream, there is a `sort` delay between the time t0 at which the photodetector subsystem 20 generates an output signal 21 for that cell and the time of the sorting pulse at which a droplet 23 containing that cell breaks off (at location 25) from the fluid stream.
Knowing the exact duration of this sort delay is critical to accurate sorting of the drops, since only the last attached droplet that breaks off from the fluid stream at the time t1 of the applied sort charging pulse 32 will be deflected by the deflection plates 33 and 35, and subsequently collected into the sorted droplet collection container 41.
Sort delay is affected by various parameters including the pressure of the carrier fluid, size and surface characteristics of the droplet generator exit port, the viscosity of the carrier fluid, and the amplitude of the piezo vibration. While some parameters, such as the pressure of the fluid carrier, which affect the position of the droplet formation point, can be controlled with precision, others cannot be controlled. For example, material may build up on the flow chamber exit port, causing a change in the natural energy of the fluid stream, and moving the droplet formation point closer to the flow chamber. Other factors include acoustic coupling of the instrument vibration, room noise, vibration in the room machinery external to the unit, and so on.
As a consequence, it is standard practice to conduct a preliminary set of test and calibration steps to accurately establish the droplet formation location 25. As a non-limiting example, this may be accomplished by initially manually setting the droplet formation point 25 at some predetermined distance from the laser intersection point 15, using a precision imaging aid (such as a microscope objective or a video camera) to observe the fluid steam. Strobing a light emitting diode in sync with the excitation frequency of the piezoelectric drive signal to the droplet generator 27 will make the droplets 23 formed from the fluid stream appear to be stationary. Then, by controllably increasing or decreasing the amplitude of the piezoelectric drive signal, the operator can move the droplet formation point closer or farther away from the laser intersection point, until the point at which the drops first form coincides with a reference or positioning mark.
Next, the operator inputs to the sorting system a sort delay time that has been determined on the basis of previous experimentation, so as to place the system within several drops of the actual sort delay time. In order to bring the system to within one droplet of accuracy, the operator sets up and runs a calibration sort operation, using test beads, which mimic biological cells in terms of size. The beads are sorted onto a slide, and the slide is observed (under a microscope) to determine whether the number of beads on the slide coincides with the number of beads the system reported as having sorted.
If the numbers do not coincide, then the system is adjusted by changing the sort delay time, or by moving the droplet formation point by varying the amplitude of the acoustic drive signal. This operation is iteratively repeated as necessary until the beads counts are correct. With the system thus initially calibrated, it may then be monitored visually for drift, with the operator observing the fluid stream and droplets for movement. To verify that the sort parameters remain the same, the slide and bead analysis sequence described above may be repeated. It will be readily appreciated that this trial and error procedure is a time consuming process, and sample may be lost or the sort container contaminated during the sorting process without operator knowledge.
Unfortunately, proposals that have been suggested to remedy the problem are complex and costly, and not necessarily complete solutions. For example, one proposal is to incorporate a test mode optical forward error correction system, comprised of an additional laser--photodetection subsystem, that takes a `second look` into the continuous fluid stream at some point downstream of the laser beam intersection location 15, but prior to the droplet break off point 25. The purpose of the second optical system is to confirm that test beads that have been injected in the fluid stream arrive at the downstream detection location at a time that they are expected.
In accordance with another proposal, an auxiliary laser is employed to determine whether there has been a shift in the overall velocity of the droplet stream. An obvious shortcoming of this approach is that it does not address the fundamental problem of determining exactly where the last attached droplet breaks off from the fluid stream. A further proposal places a second laser at an initially established droplet break off point and then monitors the stream at that point. Unfortunately, since the laser is fixedly positioned, it cannot be readily repositioned if the break off point moves.